There are known some types of viruses which cause viral hepatitis. Hepatitis A virus is an RNA virus having a diameter of 27 nm which belongs to picornavirus and causes epidemic hepatitis through oral infection (Fineston, S. M. et al., Science, 182: p. 1026, 973). Hepatitis B virus is a DNA virus having a diameter of 42 nm which belongs to hepadnavirus and causes hepatitis through blood infection (Dane, O. S. et al., Lancet. I: p. 241, 1974) For these viruses, definite diagnostic methods and prevention measures have been established.
A hepatitis virus which does not belong to any of the above types was called non-A, non-B hepatitis virus. Recently, it has been known that there are two kinds of non-A, non-B hepatitis viruses. One is calicivirus like hepatitis virus having a diameter of 27 to 32 nm and causing epidemic hepatitis through oral infection in India, Myanma, Afghanistan, north Africa and etc. (Khuroo, M. S. Am. J. Med.,68: p. 818-824, 1980). This was named Hepatitis E virus (HEV) for "E" of epidemic or enteric.
The other is a virus which causes most of hepatitis after blood transfusion and which is called hepatitis C virus. The existence of the virus has been known for a long time (Tabor, E. et al., Lancet. I: p. 463, 1978) but the virus itself has not been made sufficiently clear in spite of many efforts by researchers throughout the world.
Under such circumstances, Choo, Q. et al. of the United States succeeded in cloning of cDNA of HCV in 1989 and reported that they established a system for the detection of anti-HCV antibodies using an expressed protein encoded by a part of the cDNA (Choo, Q. et al., Science, 224, p. 359-362, 1989; Kuo, G. et al., Science 224, p. 362-364, 1989). On the other hand, the inventors of the present invention independently have succeeded in cloning of cDNA coding for HCV structural protein region using high GPT plasma of a Japanese blood donor and have filed a patent application World Intellectual Property Organization International Publication No. WO 91/04262.
An ELISA system for the detection of anti-HCV antibodies prepared by Kuo, G. et al. (HCV Ab ELISA kit manufactured by Ortho Inc.) uses as an antigen a protein which is expressed in a yeast as a fused protein between superoxide dismutase (SOD) and a part called C100 of NS3 to NS4 region which is considered nonstructural region of the virus gene. This system can detect specifically and sensitively antibodies against virus in patients infected with HCV and is highly valuable as a judging measure in screening of blood for transfusion which could spread hepatitis C after blood transfusion. However, anti-C100 antibody measured in the above-mentioned antibody detection system causes positive reaction after the symptoms of hepatitis C is shown but cannot be detected usually from 3 to 6 months after the infection, during which the system cannot be used as a diagnostic measure for hepatitis C. This is one of the biggest problems of the system. Even if only blood which is negative in anti-C100 antibody test using the system is transfused, hepatitis C breaks out to some extent. It is, therefore, considered that the antibody test using the system could detect and exclude about 50% of post-tranfusion hepatitis. Thus, there is a strong need for a new method for detecting the antibody. Further, the following problems still remain unsolved in a detection system using a fused protein virus antigen produced by a recombinant DNA technique.
1) There is a possibility of decomposition of desired expressed protein by proteases derived from a recombinant microorganism which produced the protein. PA0 2) Many and complicated processes are required to purify desired antigenic protein from a large amount of proteins derived from a cultured recombinant microorganism, supernatant of a cultured cell, or cell body. PA0 3) There is a possibility of non specific reaction caused by a fused protein or a recombinant microorganism used for the expression. PA0 4) Since it is essential to culture a recombinant microorganism, lot-to-lot variation is apt to occur.
The above-mentioned commercially available system for the detection of anti-HCV antibodies is very significant because it can detect rapidly and easily antibodies against hepatitis C virus, which has never been successful. However, there is a need for a diagnostic method with "100% accuracy" wherein a detection rate is improved and a non-specific reaction is inhibited. From the point of quality control, there is room for improvement. For these purposes, further researches and developments are now carried out.
The inventors of the present invention prepared a synthetic peptide of the structural region encoded by independently cloned cDNA and developed some systems for the detection of antibodies using the peptide so that it is possible to avoid non-specific reaction caused by a fused protein, non-specific reaction caused by a transformant used for the expression of the protein, or difficulty of quality control. As a result, they have accomplished the present invention.